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In this psycho-neuro-endocrine study the molecular basis of different variants of steroid receptors as well as highly conserved non steroidal receptors was investigated. These nuclear receptors (NRs) are important key regulators of a wide variety of different physiological and pathophysiological challenges ranging from inflammation and stress to complex behaviour and disease. NRs control gene transcription in a ligand dependent manner and are embedded in the huge interaction network of the neuroendocrine and immune system. Two receptors, the glucocorticoid receptor (GR) and the chicken ovalbumin upstream promoter-transcription factorII (Coup-TFII), both expressed in the immune and nervous system, were investigated regarding possible splice variants and their implication in the control of gene transcription. Both NRs are known to interact and modulate each other- target gene regulation. This study could be shown that both NRs have different splice variants that are expressed in a tissue specific manner. The different 5-´alternative transcript variants of the human GR were in silico identified in other species and evidence for a highly conserved and tightly controlled function was provided. Investigations of the N-terminal transactivation domain of the GR showed a deletion suggesting an altered glucocorticoid-dependent transactivation profile. The newly identified alternative transcript variant of Coup-TFII leads to a DNA binding deficient Coup-TFII isoform that is highly expressed in the brain. This Coup-TFII isoform alters Coup-TFII target gene expression and is suggested to interact with GR via its ligand binding domain resulting in an impaired GR target gene regulation in the nervous system. In this thesis it was demonstrated that NR variants are important for the understanding of the enormous regulatory potential of this receptor family and have to be taken into account for the development of therapeutic strategies for complex diseases such as stress related and neurodegenerative disorders.

There is a lot of evidence for the impact of acute glucocorticoid treatment on hippocampus-dependent explicit learning and memory (memory for facts and events). But there have been few studies, investigating the effect of glucocorticoids on implicit learning and memory. We conducted three studies with different methodology to investigate the effect of glucocorticoids on different forms of implicit learning. In Study 1, we investigated the effect of cortisol depletion on short-term habituation in 49 healthy subjects. 25 participants received oral metyrapone (1500 mg) to suppress endogenous cortisol production, while 24 controls received oral placebo. Eye blink electromyogram (EMG) responses to 105 dB acoustic startle stimuli were assessed. Effective endogenous cortisol suppression had no effect on short-term habituation of the startle reflex, but startle eye blink responses were significantly increased in the metyrapone group. The latter findings are in line with previous human studies, which have shown that excess cortisol, sufficient to fully occupy central nervous system (CNS) corticosteroid receptors, may reduce startle eye blink. This effect may be mediated by CNS mechanisms controlling cortisol feedback. In Study 2, we investigated delay or trace eyeblink conditioning in a patient group with a relative hypocortisolism (30 patients with fibromyaligia syndrome/FMS) compared to 20 healthy control subjects. Conditioned eyeblink response probability was assessed by EMG. Morning cortisol levels, ratings of depression, anxiety and psychosomatic complaints as well as general symptomatology and psychological distress were assessed. As compared to healthy controls FMS patients showed lower morning cortisol levels, and trace eyeblink conditioning was facilitated whereas delay eyeblink conditioning was reduced. Cortisol measures correlate significantly only with trace eyeblink conditioning. Our results are in line with studies of pharmacologically induced hyper- and hypocortisolism, which affected trace eyeblink conditioning. We suggest that endocrine mechanisms affecting hippocampus-mediated forms of associative learning may play a role in the generation of symptoms in these patients.rnIn Study 3, we investigated the effect of excess cortisol on implicit sequence learning in healthy subjects. Oral cortisol (30 mg) was given to 29 participants, whereas 31 control subjects received placebo. All volunteers performed a 5-choice serial reaction time task (SRTT). The reaction speed of every button-press was determined and difference-scores were calculated as a proof of learning. Compared to the control group, we found a delayed learning in the cortisol group at the very beginning of the task. This study is the first human investigation, indicating impaired implicit memory function after exogenous administration of the stress hormone cortisol. Our findings support a previous neuroimaging study, which suggested that the medial temporal lobe (including the hippocampus) is also active in implicit sequence learning, but our results may also depend on the engagement of other brain structures.

Stress is a common phenomenon for animals living in the wild, but also for humans in modern societies. Originally, the body's stress response is an adaptive reaction to a possibly life-threatening situation, and it has been shown to impact on energy distribution and metabolism, thereby increasing the chance of survival. However, stress has also been shown to impact on mating behaviour and reproductive strategies in animals and humans. This work deals with the effect of stress on reproductive behavior. Up to now, research has only focused on the effects of stress on reproduction in general. The effects of stress on reproduction may be looked at from two points of view. First, stress affects reproductive functioning by endocrine (e.g. glucocorticoid) actions on the reproductive system. However, stress can also influence reproductive behavior, i.e. mate choice and mating preferences. Animals and humans do not mate randomly, but exhibit preferences towards mating partners. One factor by which animals and humans choose their mating partners is similarity vs. dissimilarity: Similar mates usually carry more of one's own genes and the cooperation between similar mates is, at least theoretically, less hampered by expressing diverse behaviors. By mating with dissimilar mates on the other hand one may acquire new qualities for oneself, but also for one's offspring, useful to cope with environmental challenge. In humans we usually find a preference for similar mates. Due to the high costs of breeding, variables like cooperation and life-long partnerships may play a greater role than the acquaintance of new qualities.The present work focuses on stress effects on mating preferences of humans and will give a first answer to the question whether stress may affect our preference for similar mates. Stress and mating preferences are at the centre of this work. Thus, in the first Chapter I will give an introduction on stress and mating preferences and link these topics to each other. Furthermore, I will give a short summary of the studies described in Chapter II - Chapter IV and close the chapter with a general discussion of the findings and directions for further research on stress and mating preferences. Human mating behavior is complex, and many aspects of it may not relate to biology but social conventions and education. This work will not focus on those aspects but rather on cognitive and affective processing of erotic and sexually-relevant stimuli, since we assume that these aspects of mating behaviour are likely related to psychobiological stress mechanisms. Therefore, a paradigm is needed that measures such aspects of mating preferences in humans. The studies presented in Chapter II and Chapter III were performed in order to develop such a paradigm. In these studies we show that affective startle modulation may be used to indicate differences in sexual approach motivation to potential mating partners with different similarity levels to the participant. In Chapter IV, I will describe a study that aimed to investigate the effects of stress on human mating preferences. We showed that stress reverses human mating preferences: While unstressed individuals show a preference for similar mates, stressed individuals seem to prefer dissimilar mates. Overall, the studies presented in this work showed that affective startle modulation can be employed to measure mating preferences in humans and that these mating preferences are influenced by stress.

Stress and pain are common experiences in human lives. Both, the stress and the pain system have adaptive functions and try to protect the organism in case of harm and danger. However, stress and pain are two of the most challenging problems for the society and the health system. Chronic stress, as often seen in modern societies, has much impact on health and can lead to chronic stress disorders. These disorders also include a number of chronic pain syndromes. However, pain can also be regarded as a stressor itself, especially when we consider how much patients suffer from long-lasting pain and the impact of pain on life quality. In this way, the effects of stress on pain can be fostered. For the generation and manifestation of chronic pain symptoms also learning processes such as classical conditioning play an important role. Processes of classical conditioning can also be influenced by stress. These facts illustrate the complex and various interactions between the pain and the stress systems. Both systems communicate permanently with each other and help to protect the organism and to keep a homeostatic state. They have various ways of communication, for example mechanisms related to endogenous opioids, immune parameters, glucocorticoids and baroreflexes. But an overactivation of the systems, for example caused by ongoing stress, can lead to severe health problems. Therefore, it is of great importance to understand these interactions and their underlying mechanisms. The present work deals with the relationship of stress and pain. A special focus is put on stress related hypocortisolism and pain processing, stress induced hypoalgesia via baroreceptor related mechanisms and stress related cortisol effects on aversive conditioning (as a model of pain learning). This work is a contribution to the wide field of research that tries to understand the complex interactions of stress and pain. To demonstrate the variety, the selected studies highlight different aspects of these interactions. In the first chapter I will give a short introduction on the pain and the stress systems and their ways of interaction. Furthermore, I will give a short summary of the studies presented in Chapter II to V and their background. The results and their meaning for future research will be discussed in the last part of the first chapter. Chronic pain syndromes have been associated with chronic stress and alterations of the HPA axis resulting in chronic hypocortisolism. But if these alterations may play a causal role in the pathophysiology of chronic pain remains unclear. Thus, the study described in Chapter II investigated the effects of pharmacological induced hypocortisolism on pain perception. Both, the stress and the pain system are related to the cardiovascular system. Increase of blood pressure is part of the stress reaction and leads to reduced pain perception. Therefore, it is important for the usage of pain tests to keep in mind potential interferences from activation of the cardiovascular system, especially when pain inhibitory processes are investigated. For this reason we compared two commonly and interchangeably used pain tests with regard to the triggered autonomic reactions. This study is described in chapter III. Chapter IV and V deal with the role of learning processes in pain and related influences of stress. Processes of classical conditioning play an important role for symptom generation and manifestation. In both studies aversive eyeblink conditioning was used as a model for pain learning. In the study described in Chapter IV we compared classical eyeblink conditioning in healthy volunteers to patients suffering from fibromyalgia, a chronic pain disorder. Also, differences of the HPA axis, as part of the stress system, were taken in account. The study of Chapter V investigated effects of the very first stress reaction, particularly rapid non-genomic cortisol effects. Healthy volunteers received an intravenous cortisol administration immediately before the eyeblink conditioning. Rapid effects have only been demonstrated on a cellular level and on animal behavior so far. In general, the studies presented in this work may give an impression of the broad variety of possible interactions between the pain and the stress system. Furthermore, they contribute to our knowledge about theses interactions. However, more research is needed to complete the picture.

Startle reactions are fast, reflexive, and defensive responses which protect the body from injury in the face of imminent danger. The underlying reflex is basic and can be found in many species. Even though it consists of only a few synapses located in the brain stem, the startle reflex offers a valuable research method for human affective, cognitive, and psychological research. This is because of moderating effects of higher mental processes such as attention and emotion on the response magnitude: affective foreground stimulation and directed attention are validated paradigms in startle-related research. This work presents findings from three independent research studies that deal with (1) the application of the established "affective modulation of startle"-paradigm to the novel setting of attractiveness and human mating preferences, (2) the question of how different components of the startle response are affected by a physiological stressor and (3) how startle stimuli affect visual attention towards emotional stimuli. While the first two studies treat the startle response as a dependent variable by measuring its response magnitude, the third study uses startle stimuli as an experimental manipulation and investigates its potential effects on a behavioural measure. The first chapter of this thesis describes the basic mechanisms of the startle response as well as the body of research that sets the foundation of startle research in psychophysiology. It provides the rationale for the presented studies, and offers a short summary of the obtained results. Chapter two to four represent primary research articles that are published or in press. At the beginning of each chapter the contribution of all authors is explained. The references for all chapters are listed at the end of this thesis. The overall scope of this thesis is to show how the human startle response is modulated by a variety of factors, such as the attractiveness of a potential mating partner or the exposure to a stressor. In conclusion, the magnitude of the startle response can serve as a measure for such psychological states and processes. Beyond the involuntary, physiological startle reflex, startle stimuli also affect intentional behavioural responses, which we could demonstrate for eye movements in a visual attention paradigm.

The glucocorticoid (GC) cortisol, main mediator of the hypothalamic-pituitary-adrenal axis, has many implications in metabolism, stress response and the immune system. GC function is mediated mainly via the glucocorticoid receptor (GR) which binds as a transcription factor to glucocorticoid response elements (GREs). GCs are strong immunosuppressants and used to treat inflammatory and autoimmune diseases. Long-term usage can lead to several irreversible side effects which make improved understanding indispensable and warrant the adaptation of current drugs. Several large scale gene expression studies have been performed to gain insight into GC signalling. Nevertheless, studies at the proteomic level have not yet been made. The effects of cortisol on monocytes and macrophages were studied in the THP-1 cell line using 2D fluorescence difference gel electrophoresis (2D DIGE) combined with MALDI-TOF mass spectrometry. More than 50 cortisol-modulated proteins were identified which belonged to five functional groups: cytoskeleton, chaperones, immune response, metabolism, and transcription/translation. Multiple GREs were found in the promoters of their corresponding genes (+10 kb/-0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS)). High quality GREs were observed mainly in cortisol modulated genes, corroborating the proteomics results. Differential regulation of selected immune response related proteins were confirmed by qPCR and immuno-blotting. All immune response related proteins (MX1, IFIT3, SYWC, STAT3, PMSE2, PRS7) which were induced by LPS were suppressed by cortisol and belong mainly to classical interferon target genes. Mx1 has been selected for detailed expression analysis since new isoforms have been identified by proteomics. FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein and contained the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggested GC inducibility of all transcripts. 2D DIGE combined with 2D immunoblotting revealed the existence of several previously unknown FKBP51 isoforms, possibly resulting from these transcripts. Additionally multiple post-translational modifications were found, which could lead to different subcellular localization in monocytes and macrophages as seen by confocal microscopy. Similar results were obtained for the different cellular subsets of human peripheral blood mononuclear cells (PBMCs). FKBP51 was found to be constitutively phosphorylated with up to 8 phosphosites in CD19+ B lymphocytes. Differential Co-immunoprecipitation for cytoplasm and nucleus allowed us to identify new potential interaction partners. Nuclear FKBP51 was found to interact with myosin 9, whereas cytosolic FKBP51 with TRIM21 (synonym: Ro52, Sjögren`s syndrome antigen). The GR has been found to interact with THOC4 and YB1, two proteins implicated in mRNA processing and transcriptional regulation. We also applied proteomics to study rapid non-genomic effects of acute stress in a rat model. The nuclear proteome of the thymus was investigated after 15 min restraint stress and compared to the non-stressed control. Most of the identified proteins were transcriptional regulators found to be enriched in the nucleus probably to assist gene expression in an appropriate manner. The proteomic approach allowed us to further understand the cortisol mediated response in monocytes/macrophages. We identified several new target proteins, but we also found new protein variants and post-translational modifications which need further investigation. Detailed study of FKBP51 and GR indicated a complex regulation network which opened a new field of research. We identified new variants of the anti-viral response protein MX1, displaying differential expression and phosphorylation in the cellular compartments. Further, proteomics allowed us to follow the very early effects of acute stress, which happen prior to gene expression. The nuclear thymocyte proteome of restraint stressed rats revealed an active preparation for subsequent gene expression. Proteomics was successfully applied to study differential protein expression, to identify new protein variants and phosphorylation events as well as to follow translocation. New aspects for future research in the field of cortisol-mediated immune modulation have been added.

Stress represents a significant problem for Western societies inducing costs as high as 3-4 % of the European gross national products, a burden that is continually increasing (WHO Briefing, EUR/04/5047810/B6). The classical stress response system is the hypothalamic-pituitary-adrenal (HPA) axis which acts to restore homeostasis after disturbances. Two major components within the HPA axis system are the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Cortisol, released from the adrenal glands at the end of the HPA axis, binds to MRs and with a 10 fold lower affinity to GRs. Both, impairment of the HPA axis and an imbalance in the MR/GR ratio enhances the risk for infection, inflammation and stress related psychiatric disorders. Major depressive disorder (MDD) is characterised by a variety of symptoms, however, one of the most consistent findings is the hyperactivity of the HPA axis. This may be the result of lower numbers or reduced activity of GRs and MRs. The GR gene consists of multiple alternative first exons resulting in different GR mRNA transcripts whereas for the MR only two first exons are known to date. Both, the human GR promoter 1F and the homologue rat Gr promoter 1.7 seem to be susceptible to methylation during stressful early life events resulting in lower 1F/1.7 transcript levels. It was proposed that this is due to methylation of a NGFI-A binding site in both, the rat promoter 1.7 and the human promoter 1F. The research presented in this thesis was undertaken to determine the differential expression and methylation patterns of GR and MR variants in multiple areas of the limbic brain system in the healthy and depressed human brain. Furthermore, the transcriptional control of the GR transcript 1F was investigated as expression changes of this transcript were associated with MDD, childhood abuse and early life stress. The role of NGFI-A and several other transcription factors on 1F regulation was studied in vitro and the effect of Ngfi-a overexpression on the rat Gr promoter 1.7 in vivo. The susceptibility to epigenetic programming of several GR promoters was investigated in MDD. In addition, changes in methylation levels have been determined in response to a single acute stressor in rodents. Our results showed that GR and MR first exon transcripts are differentially expressed in the human brain, but this is not due to epigenetic programming. We showed that NGFI-A has no effect on endogenous 1F/1.7 expression in vitro and in vivo. We provide evidence that the transcription factor E2F1 is a major element in the transcriptional complex necessary to drive the expression of GR 1F transcripts. In rats, highly individual methylation patterns in the paraventricular nucleus of the hypothalamus (PVN) suggest that this is not related to the stressor but can rather be interpreted as pre-existing differences. In contrast, the hippocampus showed a much more uniform epigenetic status, but still is susceptible to epigenetic modification even after a single acute stress suggesting a differential "stateâ€Ÿ versus "traitâ€Ÿ regulation of the GR gene in different brain regions. The results of this thesis have given further insight in the complex transcriptional regulation of GR and MR first exons in health and disease. Epigenetic programming of GR promoters seems to be involved in early life stress and acute stress in adult rats; however, the susceptibility to methylation in response to stress seems to vary between brain regions.